1. Field of the Invention
A molecular or macromolecular structure and method of use are disclosed in which an active-nucleus is functionalized in at least a transient interaction with a target carrier to form a sensor that selectively associates with a target substrate or environment to produce a detectable signal. More specifically, a functionalized active-nucleus complex sensor is described in which an active-nucleus gas such as hyperpolarized xenon, hyperpolarized helium, or sulfur hexafluoride, or active-nuclei 19F derivatives are bound in a carrier structure having a binding region specific for a target species. Upon binding to the target species the active-nucleus produces a detectable nuclear magnetic resonance signal or is detectable as a magnetic resonance imaging contrast agent. A plurality of target specific sensors may be utilized in the assaying and screening of samples containing the plurality of targets under either in vivo or in vitro conditions.
2. Description of the Background Art
The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. Recent biosensor technologies exploit surface plasmon resonance (1), fluorescence polarization (2), and fluorescence resonance energy transfer as detection methods (3). Although the sensitivity of such techniques is excellent, it has proven challenging to extend these assays to multiplexing capabilities because of the difficulty in distinguishing signals from different binding events. While nuclear magnetic resonance (NMR) spectroscopy is able to finely resolve signals from different molecules and environments, the spectral complexity and low sensitivity of NMR spectroscopy normally preclude its use as a detector of molecular targets in complex mixtures. Notable successes (4,5) in the application of NMR to such problems are still limited by long acquisition times or a limited number of detectable analytes. Laser polarized xenon NMR benefits from good signal to noise and spectral simplicity with the added advantage of substantial chemical shift sensitivity.
U.S. Pat. No. 5,642,625 discloses a high volume hyperpolarizer for spin-polarized noble gas. A method and apparatus are presented that allow spin exchange between atoms of the noble gas and an alkali metal such as rubidium.
Described in U.S. Pat. No. 5,785,953 is a magnetic resonance imaging technique using hyperpolarized noble gases as contrast agents. In particular, hyperpolarized xenon and helium are utilized in spatial distribution studies.
The foregoing references/patents reflect the state of the art of which the applicant is aware and are tendered with the view toward discharging applicant""s acknowledged duty of candor in disclosing information which may be pertinent in the examination of this application. It is respectfully submitted, however, that none of these references/patents teach or render obvious, singly or when considered in combination, applicant""s claimed invention.
An object of the present invention is to disclose a sensor and method of use comprising an active-nucleus (guest) and target carrier (host) that generates an NMR and/or MRI detectable signal upon association with a biological target.
Another object of the present invention is to relate a biosensor and method of in vivo and in vitro assaying/screening use that comprises a functionalize active-nucleus complex that selectively binds to and signals the presence of a desired biological target species.
A further object of the present invention is to describe biosensors and methods of in vivo and in vitro assaying/screening use that comprises a plurality of functionalize active-nucleus complexes with each complex selectively binding to and signaling the presence of a desired biological target species or analyte.
Still another object of the present invention is to present a biosensor and method of use in which the biosensor comprises an active-nucleus bound to a target carrier in which when the target carrier binds to a target species/analyte a detectable signal is produced upon the binding or upon alterations in the target species/analyte or its environment after the binding.
Yet a further object of the present invention is to disclose a plurality of biosensors and a multiplexed method of use in which each of the biosensors comprises an active-nucleus bound to a target carrier in which when the target carrier binds to a target species/analyte a detectable signal is produced upon the binding or upon alterations in the target species/analyte or its environment after the binding, wherein all the biosensors"" signals are simultaneously detectable.
Disclosed is a novel, functionalized active-nucleus sensor or biosensor that is directed to and signals the presence of a desired biological target species, often of biological origin or importance. An active-nucleus that presents a detectable signal to either nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) techniques is utilized in conjunction with a target specific carrier that interacts with both the active-nucleus and a biological target substrate or environment. The active-nucleus is capable of at least a minimal transient binding to a targeting carrier. The targeting carrier associates with the target substrate or environment, thereby stimulating the production of or change in the detectable signal from the active-nucleus in a functionalized interaction. xe2x80x9cFunctionalizedxe2x80x9d implies that when the active-nucleus is bound, in at least a minimal transient manner, by the targeting carrier, that the active-nucleus then responds to and signals the association between the targeting carrier and the target substrate or environment.
Since the basic subject invention enables the creation of several extremely powerful and versatile sensors and techniques that have eluded researchers for many years, a number of related embodiments are disclosed below. One requirement for the subject invention is that the reporter nucleus be sufficiently xe2x80x9cactivexe2x80x9d or capable of producing a signal that is detectable by NMR or MRI techniques. Hyperpolarized noble gases such as xenon and helium meet this requirement, as do other nuclei such as 19F, if present in sufficiently high concentrations. Thus, xe2x80x9cactivexe2x80x9d implies that the nucleus generates a suitable signal that is capable of detection by NMR (either in strong or weak magnetic fields) and/or MRI contrast procedures. Several relatively standard techniques now exist for hyperpolarizing noble gases and include optical pumping or spin exchange procedures.
It is important to appreciate that for the subject invention the signal produced by the functionalized active-nucleus is studied directly to follow the behavior of the biological target substrate or environment. For example, xenon (as indicated above, other suitable active-nuclei are also contemplated as being within the realm of this disclosure), has a chemical shift that is enormously sensitive to its local chemical environment. With the large xenon NMR signal created by optical pumping, the chemical shift can easily serve as a signature for the different chemical surroundings in which the xenon is found. Direct interaction between xenon and a target molecule has been observed by measuring the chemical shift and relaxation properties on xenon (in particular see, S. M. Rubin, M. M. Spence, B. M. Goodson, D. E. Wemmer, A. Pines, Proceedings of the National Academy of Sciences of the United States of America 97, 9472-9475 (2000) that was part of the Provisional Application to which this application claims priority). However, the observation is of this direct contact may be limited by the weak binding of xenon (or other suitable active-nuclei) to many target molecules of interest. To enhance the binding of the xenon, for example, to the biological target species/substrate/molecule/analyte of interest, and thus the population of xenon in contact with the target species/substrate/molecule/analyte, the xenon can be functionalized to strongly bind to the biological target species/substrate/molecule/analyte. This can be achieved by placing the xenon, or other suitable active-nuclei, in a target carrier that chemically recognizes and binds to the target. The target carrier has a first binding region that binds the xenon for at least a minimal transient period (xe2x80x9cminimalxe2x80x9d in the sense of a sufficiently long period of time to produce a useful signal) or, preferably, very strongly binds the xenon, and can not itself quickly relax the xenon polarization. Amplification of the sensing for both xenon and helium may be achieved by utilizing a xe2x80x9cpoolxe2x80x9d of hyperpolarized active-nuclei atoms that either sense the environment by changes in the functionalized active-nucleus carrier complex (molecule, supramolecular, or microbubble environment) or else are in sufficiently rapid chemical exchange with active-nuclei in biosensor sites that are so sensitive, thereby amplifying the detection intensity yet further.
The target carrier has a second binding region that binds to or reacts with the target species/substrate/molecule/analyte. The target carrier allows xenon, or other active-nuclei, to be held in close proximity to the desired target, giving rise to a signal at a distinctive frequency indicating the presence of the target species/substrate/molecule/analyte. The functionalized active-nucleus/target carrier complex can xe2x80x9crecognizexe2x80x9d any one of a wide variety of biological target species/substrates/molecules/analytes (virtually an unlimited set of organic/biomolecular structures) including biologically important species such as proteins, nucleic acids, carbohydrates, lipids, metabolites, and the like in either an in vitro or non-invasive in vivo setting at either high or low NMR utilized field strengths. For example, with diseases, the diagnostic power of the subject invention is quite clear. Various diseases present characteristic/defining targets such as unusual membrane proteins, lipids, or carbohydrates, unusual analytes in body fluids, and the like whose presence can easily be detected with the subject invention.
The subject method of assaying and screening for target species in in vivo and in vitro samples/subjects has many strengths, including the large signal to noise ratio afforded by the high polarization achieve with hyperpolarization of xenon, helium, and other suitable nuclei. With xenon, for example, there is a negligible natural presence of xenon, so there would be no interference from background xenon signals. In contrast to fluorescence (and other techniques that generate overlapping or interfering detection signals) assays and screening procedures, multiple functionalized active-nuclei tests are possible in one system (test-tube, plate, microplate, and the like), by creating active-nuclei carriers targeting different species/substrates/molecules/analytes or by altering the structure of the probe itself or both (see below). Each target would give rise to a separate active-nucleus chemical shift. These assays and screenings could also be carried out non-invasively in vivo, avoiding the exposure to radiation that radiometric assays and screenings require. In the case of optical pumping to create hyperpolarization of xenon and helium, because the large active-nuclei signals are generated by the optical pumping, high magnetic fields are unnecessary (as mentioned previously), and the chemical shifts can be detected in low magnetic fields using a superconducting quantum interference device (SQUID).
A preliminary calculation was performed (verified by the results obtained in Experimental Example #1, found below) to explore the initial feasibility of the subject technique in vivo. Based on capabilities of current spectroscopy, 200 nanomoles of nuclear spins are necessary to measure a signal. To compete with or match other forms of assays or screening procedures, 20 picomoles of target species must be detectable, A factor of 104 in signal is required. The hyperpolarization compensates for at least a factor of 103, and the additional factor of ten is gained by the relatively simplicity of the spectrum, contrasted with a target (protein and the like) spectrum.
In its most basic configuration the subject invention comprises an active-nucleus and a target carrier that associates with both the active-nucleus and a desired target species to produce an detectable characteristic signal (typically a chemical shift or relaxation time for NMR or a contrast capability for MRI). The functionalized active-nucleus complex or subject biosensor that may comprise one or more identical or varied second binding regions. Additionally, the functionalized active-nucleus complex or subject biosensor may have varied first binding regions. Also, both the first and second binding regions could be varied within the same subject biosensor. As indicated, the subject invention allows a huge array of possible target species/substrates/molecules/analytes to be assayed/screened for in a parallel or multiplexing detection style within a single sample/subject.
Several possible active-nuclei gases exist, preferable hyperpolarized xenon and hyperpolarized helium, however, 19F and similar nuclei, in sufficient concentration, are also contemplated. With fluorine atoms, an exemplary functionalized sensor comprises a target carrier having multiple fluorines such as a polyfluorinated dendrimer that selectively binds an organic target species/substrate/molecule/analyte or a form of fluorine such as sulfur hexafluoride trapped/bound within a functionalized (target specific binding) enclosing structure such as in xe2x80x9cbubblexe2x80x9d or xe2x80x9cmicrobubblexe2x80x9d environment as exemplified by a liposome, micelle, vesicle, bucky-ball type structures, natural and synthetic polymeric cages, and like. Conformational changes or alterations in the effective pressure on the xe2x80x9cbubblexe2x80x9d or xe2x80x9cmicrobubblexe2x80x9d would induce detectable signal variations from the subject biosensor. Variations in the immediate vicinity or environment of the biosensor should be detectable and include changes in ion concentrations, functioning of an ion channel, oxygen levels/distribution, neuron activity, and the like. It is noted that hyperpolarized xenon and hyperpolarized helium will also function as the signal reporting active-nuclei within similar functionalized xe2x80x9cbubblexe2x80x9d or xe2x80x9cmicrobubblexe2x80x9d structures.
The first binding region of the targeting carrier interacts/associates/binds with the active-nucleus. This first binding region includes structures such as monoclonal antibodies, dendrimers, self-assembled lipid complexes, liposomes, cyclodextrins, cryptands, carcerands, microbubbles, micelles, vesicles, molecular tennis balls, fullerenes, many general cage-like structures, and the like.
The second binding region in the targeting carrier comprises that portion of the subject biosensor that interacts with the target species/substrate/molecule/analyte. It is noted that multiple second binding regions are contemplated and may be identical or varied for attachment to a plurality of target sites.
The basic subject biosensor may contain additional useful components/structures. One or more xe2x80x9ctetherxe2x80x9d regions may be included and serve to separate the first and second binding regions and to permit a region that may be further derivatized with additional moieties such as solubilizing regions. The solubilizing regions may contain polypeptides, carbohydrates, and other species that aid in solubilizing the subject probe.
More specifically, a functionalized active-nucleus biosensor is disclosed that capitalizes on the enhanced signal to noise, spectral simplicity, and chemical shift sensitivity of suitable active-nuclei gases (for example only and not by way of limitation, hyperpolarized xenon, hyperpolarized helium, and sulfur hexafluoride) and polyfluorinated containing species (utilized to target organic molecules) to detect specific targets. One subject sensor embodiment utilizes laser polarized xenon xe2x80x9cfunctionalizedxe2x80x9d by a biotin-modified supramolecular cage, including a tether region having a solubilizing region, to detect biotin-avidin binding. This biosensor methodology can be used in analyte assays and screening procedures or extended to multiplexing assays for multiple analytes of screenings for multiple species.
Other objects, advantages, and novel features of the present invention will become apparent from the detailed description that follows, when considered in conjunction with the associated drawings.